Poxviruses, including the causative agent of smallpox, Variola virus (VARV), are highly pathogenic double stranded (ds) DNA viruses. It is estimated that smallpox has caused more than 300 million deaths in the 20th century alone. Even though traditional vaccination programs have eradicated VARV as a natural pathogen, it remains that enhancing the knowledge of mechanisms of its infections and/or protection may be essential given the scenarios of zoonotic poxvirus infections (e.g. monkeypox), the re-emergence of VARV by accidental release, or the possibility of terrorist attacks with poxviruses.
The genus Orthopoxvirus contains several related viruses based on genetic similarity and immunological cross-reactivity, including VARV, the causative agent of human smallpox, ectromelia virus (ECTV) causing mousepox, cowpox virus (CPXV), monkeypox virus (MPXV), camelpox virus (CMPV), and vaccinia virus (VACV). Among these ECTV, MPXV, and VACV are used in animals as model infections for human smallpox. A number of VACV strains have been used in mice and large amounts of evidence on the immune responses induced and on the immune suppressing mechanisms employed by poxviruses have been elucidated with VACV. However, VACV is not a natural pathogen of mice and high doses are needed to lethally infect mice, even though mouse-adapted strains like VACV Western Reserve (WR) are commonly used (Williamson et al., J. Gen. Virol. 71:2761-2767 (1990)). MPXV in monkeys has the advantage that monkeys are evolutionarily much closer to humans. However, as with the VACV model in mice, non-physiological high viral doses are needed to lethally infect monkeys. Therefore, both animal models are regarded to reflect more the late stage of a VARV infection in humans (Fenner, F., Henderson, D. A., Arita, I., Jezek, Z., & Ladnyi, I. D. Smallpox and its eradication. Geneva: World Health Organization (1988); Mortimer, P. P. Clin. Infect. Dis. 36, 622-629 (2003)). Among the orthopoxvirus infection models, ECTV infection of mice stands out because it is a species-specific virus infecting its natural host and can cause fatal outcomes after inoculation with low virus doses, features that have also been described in VARV infection of humans (Fenner et al., 1988; Esteban, D. J., and Buller, R. M., J. Gen. Virol. 86:2645-2659 (2005)). For these reasons, this model is the closest model to human infection by VARV.
Pathogens are detected by the immune system via pattern recognition receptors (PRR). Among the latter is the family of Toll-like receptors (TLR). TLR7, and TLR8 and 9 recognize the nucleic acids RNA and DNA, respectively (Hemmi, H. et al., Nature 408, 740-745 (2000); Diebold, S. S. et al. Science 303, 1529-1531 (2004); Heil, F. Science 303, 1526-1529 (2004)). Double stranded DNA (dsDNA) viruses, like herpesviruses or adenoviruses, can be detected via TLR9-dependent pathways (Basner-Tschakarjan, E. et al., J. Gene Med. 8, 1300-1306 (2006); Lund, J. et al., J. Exp. Med. 198, 513-520 (2003); Krug, A. et al. Blood 103, 1433-1437 (2004); Hochrein, H. et al. Proc. Natl. Acad. Sci. U.S.A. 101, 11416-11421 (2004); Tabeta, K. et al. Proc. Natl. Acad. Sci. U.S.A. 101, 3516-3521 (2004)). However, potent alternative recognition pathways exist, possibly explaining why previous viral infection studies have demonstrated no or only mild increases of susceptibility in the absence of TLR9 (Hochrein, H. et al. Proc. Natl. Acad. Sci. U.S.A. 101, 11416-11421 (2004); Krug, A et al. Blood 103, 1433-1437 (2004); Zhu, J. et al. Blood 109, 619-625 (2007); Delale, T. J. Immunol. 175, 6723-6732 (2005); Tabeta, K. et al. Proc. Natl. Acad. Sci. U.S.A. 101, 3516-3521 (2004)).
Whereas many TLR are located at the outer membrane of the cell to monitor the extracellular space for danger signals like bacterial cell wall components, a group of TLR consisting of TLR 3, 7, 8, and 9 are associated with the endosome and monitor the endosomal lumen for nucleic acids (Wagner, H., and Bauer, S., J. Exp. Med. 203:265-268 (2006)). The TLR 3, 7, and 8 recognize RNA, whereas TLR9 recognizes DNA (Diebold et al., Science 303: 1529-153 (2004); Heil et al., Science 303:1526-1529 (2004); Hemmi et al., Nature 408:740-745 (2000); Alexopoulou et al., Nature 413:732-738 (2001)).
Expression of TLR9 differs within species. Whereas in humans B-cells and plasmacytoid DC (pDC), but not conventional DC (cDC), express and respond to TLR9 stimulation, TLR9 expression in mice is less restricted. Besides B-cells and pDC, mouse cDC and even macrophages are known to express TLR9 and respond to TLR9 ligation (Hochrein et al., Hum. Immunol. 63:1103-1110 (2002)). The natural ligand for TLR9 was originally defined to be genomic bacterial DNA, whereas oligonucleotides containing unmethylated CpG motifs adjoined by species specific motifs and often phosphorothioate-stabilized (CpG-ODN), were established as artificial ligands for TLR9 (Hemmi et al., 2000; Bauer et al., Proc. Natl. Acad. Sci. U.S. A 98:9237-9242 (2001)).
Meanwhile, the list of CpG containing natural and artificial ligands has increased to bacterial plasmid DNA and several types of CpG-ODN with differences in their chemical composition, as well as drastic differences in biological effects including IFN-I inducing capacity (Spies et al., J. Immunol. 171:5908-5912 (2003); Krieg, A. M. Nat. Rev. Drug Discov. 5:471-484 (2006)). Under conditions of enhanced uptake, non CpG-containing or fully methylated DNA as well as vertebrate DNA have also been shown to act as TLR9 agonists (Yasuda et al., J. Immunol. 174:6129-6136 (2005); Means et al., J. Clin. Invest 115:407-417 (2005)).
Poxviruses have evolved multiple strategies for immune suppression, substantiated by the fact that poxvirus genomes encode numerous molecules with immunosuppressive function. Among these are soluble cytokine and chemokine receptors and a multitude of molecules that interfere with intracellular signaling cascades (Seet, B. T. et al. Annu. Rev. Immunol. 21, 377-423 (2003)). Recently, molecules expressed by poxviruses have been shown to target members of the TLR signaling cascade, suggesting a role for TLR-dependent recognition pathways for poxviruses (Bowie, A. et al. Proc. Natl. Acad. Sci. U.S.A. 97, 10162-10167 (2000)). In fact, a role for TLR2 in the recognition of vaccinia viruses (VACV) was proposed (Zhu, J. et al. Blood 109, 619-625 (2007)).
Modified Vaccinia Ankara (MVA) virus is related to Vaccinia virus, a member of the genera Orthopoxvirus in the family of Poxviridae. MVA has been generated by 516 serial passages on chicken embryo fibroblasts of the Ankara strain of vaccinia virus (CVA) (for review see Mayr, A., et al. Infection 3, 6-14 (1975)). As a consequence of these long-term passages, the resulting MVA virus deleted about 31 kilobases of its genomic sequence and, therefore, was described as highly host cell restricted to avian cells (Meyer, H. et al., J. Gen. Virol. 72, 1031-1038 (1991)). It was shown, in a variety of animal models, that the resulting MVA was significantly avirulent (Mayr, A. & Danner, K. Dev. Biol. Stand. 41: 225-34 (1978)). Additionally, this MVA strain has been tested in clinical trials as vaccine to immunize against the human smallpox disease (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167, 375-390 (1987), Stickl et al., Dtsch. Med. Wschr. 99, 2386-2392 (1974)). These studies involved over 120,000 humans, including high risk patients, and proved that, compared to Vaccinia based vaccines, MVA had diminished virulence or infectiousness while it maintained good immunogenicity. In the decades that followed, MVA has been engineered to use it as viral vector for recombinant gene expression and as a recombinant vaccine (Sutter, G. et al., Vaccine 12: 1032-40 (1994)).
In this respect, it is most astonishing that, even though Mayr et al. demonstrated during the 1970s that MVA is highly attenuated and avirulent in humans and mammals, some recently reported observations (Blanchard et al. J. Gen. Virol. 79, 1159-1167 (1998); Carroll & Moss, Virology 238, 198-211 (1997); U.S. Pat. No. 5,185,146; Ambrosini et al., J. Neurosci. Res. 55(5), 569 (1999)) have shown that MVA is not fully attenuated in mammalian and human cell lines since residual replication might occur in these cells. It is assumed that the results reported in these publications have been obtained with various strains of MVA, since the viruses used essentially differ in their properties, particularly in their growth behavior in various cell lines.
Growth behavior is recognized as one of several indicators for virus attenuation. Generally, a virus strain is regarded as attenuated if it has lost its capacity or only has reduced capacity to reproductively replicate in host cells. The above-mentioned observation, that MVA is not completely replication incompetent in human and mammalian cells, brings into question the absolute safety of MVA as a human vaccine or a vector for recombinant vaccines.
Particularly, for a vaccine as well as for a recombinant vaccine, the balance between the efficacy and the safety of the vaccine vector virus is extremely important.
As described in WO publication 02/42480, novel MVA strains with enhanced safety have been developed. These strains are characterized by having at least one of the following advantageous properties:                (i) capability of reproductive replication in vitro in chicken embryo fibroblasts (CEF), but no capability of reproductive replication in a human cell line, as in the human keratinocyte cell line HaCaT, the human embryo kidney cell line 293, the human bone osteosarcoma cell line 143B, and the human cervix adenocarcinoma cell line HeLa;        (ii) failure to replicate in a mouse model that is incapable of producing mature B and T cells and as such is severely immune compromised and highly susceptible to a replicating virus; and        (iii) induction of at least the same level of specific immune response in vaccinia virus prime/vaccinia virus boost regimes when compared to DNA-prime/vaccinia virus boost regimes.        
One of the developed strains has been deposited at the European Collection of Animal Cell Cultures (ECACC), Vaccine Research and Production Laboratory, Public Health Laboratory Service, Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 0JG, United Kingdom, with the deposit number V00083008. This strain is referred to as “MVA-BN” throughout the specification of WO 02/42480.
The terms “not capable of reproductive replication” or “replication incompetent” mean that the virus shows an amplification ratio of less than 1 in human cell lines, such as the cell lines 293 (ECACC No. 85120602), 143B (ECACC No. 91112502), HeLa (ATCC No. CCL-2) and HaCat (Boukamp et al., J. Cell Biol. 106(3): 761-71 (1988)), under the conditions as outlined in Example 1 of WO 02/42480 for some specific MVA strains.
According to WO 02/42480, “failure to replicate in vivo” refers to viruses that do not replicate in humans and in the mice model as described in the WO 02/42480 publication.
There have been numerous reports suggesting that prime/boost regimes using MVA as a delivery vector induce poor immune responses and are inferior to DNA-prime/MVA-boost regimes (Schneider et al., Nat. Med. 4; 397-402 (1998)). In all these studies, MVA strains have been used that are different from the vaccinia viruses as developed according to WO 02/42480. As an explanation for the poor immune response obtained when MVA was used for prime and boost administration, it has been hypothesized that antibodies generated to MVA during the prime-administration neutralize the MVA given in the second immunization, preventing an effective boost of the immune response. In contrast, DNA-prime/MVA-boost regimes are reported to be superior at generating high avidity responses, because this regime combines the ability of DNA to effectively prime the immune response with the properties of MVA to boost this response in the absence of a pre-existing immunity to MVA. Clearly, if a pre-existing immunity to MVA and/or vaccinia prevents boosting of the immune response, then the use of MVA as a vaccine or therapeutic would have limited efficacy, particularly in the individuals that have been vaccinated against smallpox. However, the vaccinia virus strains according to WO 02/42480, as well as corresponding recombinant viruses harbouring heterologous sequences, can be used to efficiently first prime and then boost immune responses in native animals as well as in animals with a pre-existing immunity to poxviruses. Thus, the developed strains as described in WO 02/42480 induce at least substantially the same level of immunity in vaccinia virus prime/vaccinia virus boost regimes compared to DNA-prime/vaccinia virus boost regimes.
A vaccinia virus is regarded as inducing at least substantially the same level of immunity in vaccinia virus prime/vaccinia virus boost regimes when compared to DNA-prime/vaccinia virus boost regimes if the CTL response as measured in one of the two, or even in both assays, as described in WO 02/42480 is at least substantially the same in vaccinia virus prime/vaccinia virus boost regimes when compared to DNA-prime/vaccinia virus boost regimes.
The growth behavior of the vaccinia viruses developed according to WO 02/42480, in particular the growth behavior of MVA-BN®, indicates that the strains are far superior to any other so far characterized MVA isolate regarding attenuation in human cell lines and failure of in vivo replication. The strains are therefore ideal candidates for the development of safer products such as vaccines or pharmaceuticals.
An immune response is raised by the immune system when a foreign substance or microorganism enters the organism. By definition, the immune response is divided into a specific and an unspecific reaction, although both are closely cross linked. The unspecific immune response is the immediate defence against a wide variety of foreign substances and infectious agents. The specific immune response is the defence raised after a lag phase, when the organism is challenged with a substance for the first time. The specific immune response is highly efficient, and is responsible for the fact that an individual who recovers from a specific infection is protected against this specific infection. Thus, a second infection with the same or a very similar infectious agent causes much milder symptoms or no symptoms at all, since there is already a “pre-existing immunity” to this agent. Such immunity and the immunological memory persist for a long time, in some cases even lifelong. Accordingly, the induction of an immunological memory can result from vaccination.
The “immune system” means a complex organ involved in the defence of the organism against foreign substances and micro-organisms. The immune system comprises a cellular part comprising several cell types, such as, e.g., lymphocytes and other cells derived from white blood cells, and a humoral part comprising small peptides and complement factors.
Traditional vaccination strategies are able to induce effective and long lasting protection by inducing adaptive immune responses (antibodies, CTL). However, substantial protection can only be achieved after several days to months, optimally with a boost regime, which leaves the individual susceptible to infection during that time.
MVA is a non-replicating virus in humans, which can be administered to people with various degrees of immune deviation (HIV, allergies, atopic dermatitis, certain drug treatments), even via systemic application routes. In these cases of immune deviation, a specialized anti-viral immune cell population (pDC) is reduced in number or affected in its functional properties, which may increase the risk for viral infection.
The current view of protection against deadly poxviruses is via vaccinations. For these approaches, individuals are exposed to an attenuated (less pathogenic) poxvirus before the potential exposure to a pathogenic poxvirus. Vaccination induces adaptive immune responses like Killer T cells (CTL) and antibodies and a memory against the related vaccinating virus. This results in some reactivity against the pathogenic virus, leading to protection and quick resurrection of the memory responses upon repeated exposure. However, adaptive immune responses need time to develop, and are optimal after boosting the immune response with repetitive application of the vaccinating virus.
Recently, it was reported that, employing MVA as vaccinating virus several days (at latest 2 days) before exposure with the Vaccinia virus Western Reserve strain (VV-WR), some protection can be achieved (WO2006/089690, which is hereby incorporated by reference in its entirety). Similar results have been published by another group, which further demonstrated post-exposure treatment failed to protect animals. (Staib, C. et al. J. Gen. Virol 87, 2917-2921 (2006)). The protection levels were 1×LD50 if vaccinated 2 days before exposure to VV-WR and 12.5×LD50 if vaccinated 3 days before exposure to VV-WR. (Id.)
Stittelaar et al., Nature 439:745-748 (2006) compared the effects of antiviral treatment and smallpox vaccination upon lethal monkeypox virus infection. They reported that when monkeys were vaccinated 24 h after monkeypox virus infection, using a standard human dose of a currently recommended smallpox vaccine (Elstree-RIVM), no significant reduction in mortality was observed.
Thus, there is a need in the art for reagents and methods for immediate protection against pathogens, such as smallpox.